SUMMARY OF WORK The ATP-dependent ion pumps, which convert the chemical energy of ATP into osmotic work, are widely distributed throughout nature. In eukaryotic cells, these pumps generate transmembrane ion gradients that regulate cell shape and electrical excitability and serve as a driving force for solute transport, while in prokaryotic cells they often have a specialized role in cation homeostasis. The Na, K-ATPase in mammalian cell membranes accomplishes vectorial Na+ and K+ transport by coupling ATP hydrolysis to a sequence of conformational events that alternately expose the transport sites to opposite sides of the bilayer. Transport of cations across the bilayer leads to charge translocation which can be detected by electrical measuring circuits and potential-sensitive dyes, yielding dynamic information about the coupled conformational reactions. Using stopped-flow mixing and the potential-sensitive dye, RH 421, we measured the overall rate through several steps in the transport mechanism including ATP binding, phosphorylation, the E1P to E2P conformational transition and Na+ release (electrogenic step producing the dye signal). At saturating ATP, the kinetics of the RH 421 signal, 180 s-1, closely approximated the rate of phosphorylation, 195 s-1, determined by quenched-flow mixing, requiring that the intervening conformational transition, E1P to E2P, take place rapidly (>600 s-1 at 24 degreesC). Micromolar RH 421, which inhibits overall Na,K-ATPase activity, increased the level of phosphorylation, but not its rate, consistent with inhibition of E2P hydrolysis or Na+ release. Evidently, the delocalized positive charge on RH 421, which senses charge movement in the membrane, can also block cation translocation by electrostatic repulsion. The prokaryotic Kdp-ATPase, which maintains physiologic cytoplasmic K+ levels in E. coli, is a an oligomeric transport enzyme. This enzyme is up-regulated under conditions of K+ starvation, providing large amounts of pump protein (wild- type and mutagenized) for high-resolution kinetic studies. We used rapid quenched-flow mixing to characterize the phosphorylation dephosphorylation reactions in the vesicular and detergent-purified Kdp-ATPases. Preincubation with K+ had no effect on the rate of phosphorylation, but reduced its level consistent with increased phosphoenzyme turnover. Addition of K+ after phosphorylation did not accelerate dephosphorylation, suggesting that transport sites on the enzyme are only transiently available to bind K+. Both preparations exhibited complex kinetic behavior compatible with oligomeric catalytic subunit interactions (e.g. biphasic phosphorylation, E1P formation delayed with respect to E2P). The presence of similar kinetic behavior in the mammalian Na,K- and SR Ca-ATPases argues for an oligomeric transport mechanism in these eukaryotic enzymes.